Pharmaceutical mixture for hepatitis treatment and its preparation method

ABSTRACT

A pharmaceutical mixture for the treatment of hepatitis and its preparation method are disclosed. The method includes the following steps: pulverize the plants, macerate and extract the plant with water, concentrate the aqueous extract as the first concentrate; add ethanol to form a precipitate, collect and concentrate the liquid phase to form the second concentrate, and dry it; pass the second concentrate through the resin, elute with water, water-ethanol mixture and ethanol, collect and concentrate the water-ethanol and ethanol elution fraction as the third concentrate, and dry it. The plants in the present invention are  Boehmeria frutescens  Thunberg,  Boehmeria nivea  or the nettle family.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a pharmaceutical mixture for hepatitistreatment and its preparation method, especially to a Boehmeria familyplant for the treatment of hepatitis and preparation method of the same.

2. Description of Related Art

Chronic liver diseases (such as chronic hepatitis, cirrhosis, and livercancer) have remained significant medical problems. Liver diseasesinclude viral liver disease, alcoholic liver disease, drug or toxicantliver disease, and metabolism disorder liver disease. It is estimatedthat worldwide there are about 350 million people who are chronic B typehepatitis carriers, and 2.7 million people who are chronic C typehepatitis carriers. In Taiwan, the B type hepatitis carrying rate isabout 15 to 20%, and the C type hepatitis carrying rate is about 2 to4%.

On one hand, the current medicines for treating hepatitis, such as liverprotecting drugs, antiviral drugs or immune regulators, certainly havecurative effects but on the other hand, they have side effects and areexpensive. For example, interleukins and Lamivudine® are used fortreating B type hepatitis. The interleukins, which were approved by theU.S. FDA in 1992 for treating B type hepatitis, have only 20% positiveresponse but patients experience severe side effects. Lamivudine®, whichwas approved by the FDA in 1998 for treating B type hepatitis, also hasonly a 17 to 33% positive response. Furthermore, Lamivudine® will easilycause the mutation of B type hepatitis virus, and thus its potency isreduced.

In Chinese communities, hepatitis is usually treated with a traditionalprescription, i.e. a herbal-type medicine. The potency of that medicine,however, is not so good and has low reproducibility (probably due tolack of precise process control). Hence, there are still some problemsthat need to be overcome in the treatment of hepatitis.

The present invention provides a composition for effective treatment ofhepatitis and a novel process of the same. Liver protection can beachieved in those drug-based or other chemical (alcoholic liver disease)hepatitis cases as well as the chronic hepatitis patients.

Traditional Chinese medicines are usually extracted by water extraction,but this method cannot obtain enough active components. In addition, theactive components will lose their activity at a high extractiontemperature. Many processes in preparing pharmaceutical mixtures forhepatitis treatment have been disclosed. However, the prior arts usecomplex materials and traditional processes, which have not been able tosolve the above problems.

Alternatively, a process that utilizes organic solvents, such asmethanol, acetone, and chloroform, to extract the active components, hasbeen disclosed. These organic solvents are toxic and need to be removedcompletely before a patient can be treated. Therefore, it is desirableto provide a novel pharmaceutical mixture for hepatitis treatment andits preparation method.

In the present invention, it is observed that Boehmeria family plant iseffective for treating hepatitis. However, from the above description,the efficacy of traditional oral extract is limited. Moreover, in theindustry, the toxic organic solvents are usually adopted in theextraction process and the active component may decay. Therefore, it isdesirable to provide a novel preparation process and Boehmeria familyplant for treating hepatitis, and the components of the product are moreefficacious than traditional ones. There are more active substances andbetter curative effects, but no toxic organic solvents are used in theprocess so as to promote drug safety.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a pharmaceuticalmixture for the treatment of hepatitis and its preparation method, whichpossesses the property of liver protection, and further serves as a drugfor treating chronic hepatitis.

Another object of the present invention is to provide a pharmaceuticalmixture for the treatment of hepatitis and its preparation method,wherein the active components are extracted from the composition withoutusing of toxic organic solvents.

To achieve the object, the processes for preparing a pharmaceuticalmixture for the treatment of hepatitis in the present invention are asfollows: the plant is pulverized, macerated and extracted with water,and the aqueous extract is concentrated into a first concentrate;ethanol is added to form a precipitate, the liquid phase is collectedand concentrated to form a second concentrate, and dried; wherein theplants are Boehmeria frutescens Thunberg, Boehmeria nivea or the nettlefamily.

The process for preparing a pharmaceutical mixture for the treatment ofhepatitis in the present invention can further include: the secondconcentrate is passed through a resin, eluted with water, water-ethanolmixture and ethanol, the water-ethanol and ethanol elution fraction arecollected and concentrated as a third concentrate, and dried.

The present invention also comprises a pharmaceutical mixture for thetreatment of hepatitis according to the above-mentioned method. Thecomponents are extracted from Boehmeria frutescens Thunberg, Boehmerianivea or the nettle family.

In the present pharmaceutical mixture for the treatment of hepatitis andits preparation method, the time for maceration has no limit, butpreferably is 8-24 hrs; the extracting step further comprises a processof boiling and stirring for at least once, and each extract is collectedto form the aqueous decoction; the first decoction is preferablyconcentrated to form the first concentrate of 1-50 wt % solid content;the preferred concentration of ethanol used for precipitation is 95 wt%, and the more preferred final concentration is 40 to 80 wt %; theseparation of solid and liquid phase has no limit, and preferably isseparated by centrifugation; and the preferred solid content of thesecond concentrate is 5-50 wt %.

In the further purification process of the present pharmaceuticalmixture for treatment of hepatitis, stirring time and temperature areboth without limit, and preferably are 0.5-5 hr and 15-60° C., theprocess for solid and liquid phase separation has no limit, andpreferably is separated by centrifugation; the resin has no limit, andpreferably is macroporous resin or ion exchange resin; ethanolconcentration in the product elution is without limit, and preferably is95-100% ethanol; the ratio of water-ethanol mixture is without limit,and preferably is 1:4 to 4:1; the third concentrate is preferablyconcentrated to 5-50 wt % solid content.

In the process of the present pharmaceutical mixture for the treatmentof hepatitis, product drying has no limit, and preferably is dried bylyophilizing, spray and granulating drying, or fluidized-bed drying; andthe process of the present invention further selectively dries,pulverizes, granulates and encapsulates the final product.

Other objects, advantages, and novel features of the invention willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 1 is the relationship of product purification factors.

Table 2 shows the effect of Boehmeria frutescens Thunberg extract (JM)on the d-galactosamine-induced acute hepatitis in rats.

Table 3 shows the effect of Boehmeria frutescens Thunberg extract(BMEC-1) on the d-galactosamine-induced acute hepatitis in rats.

Table 4 shows the effects of different batches of Boehmeria frutescensThunberg extract (BMEC-1) and dose dependence effect on thed-galactosamine-induced acute hepatitis in rats.

Table 5 is the effect of Boehmeria frutescens Thunberg extract(BMEC-101) prepared from further purification process on thed-galactosamine-induced acute hepatitis in rats.

Table 6 is the report of the histopathological sections.

FIG. 1 is the histopathological sections.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Embodiment 1Preparation of Boehmeria frutescens Thunberg Extract—JM

A 0.36 kg of Boehmeria frutescens Thunberg root is macerated in 3.6 kgwater for 2 hr. After extraction at 1001° C. for about 2 hr, the mixtureis filtrated to form a decoction. Further, 2.2 kg of water is added forextraction at 100° C. for about 2 hr to form another decoction. The twodecoctions are mixed and concentrated under reduced pressure to form a0.218 kg concentrate with 15.3 wt % solid content. After lyophilization,the product JM is 33 g in weight.

Embodiment 2 Preparation of Partially Purified Boehmeria frutescensThunberg Extract—BMEC-1

A 15 kg of Boehmeria frutescens Thunberg root is macerated in 150 kgwater for 8-16 hr. After extraction at 100° C. for 2 hr. the mixture isfiltrated to form the first decoction. Further, 150 kg of water is addedfor extraction at 100° C. for 2 hr to form the second decoction. The twodecoctions are mixed into a 266 kg of decoction and concentrated underreduced pressure to form an 8.3 kg concentrate with 17.6 wt % solidcontent. The concentrate is stirred, and 15 L of 95% ethanol is addedfor ethanol precipitation by using a peristaltic pump. Finally, theethanol precipitate is separated by a centrifuge, and the supernatant iscollected, concentrated under reduced pressure, and lyophilized to formthe product BMEC-1 with 1.013 kg in weight.

Embodiment 3 Preparation of Boehmeria frutescens ThunbergExtract—BMEC-101

A 95 kg of Boehmeria frutescens Thunberg root is macerated in the 800 kgwater for 8-16 hr. After extraction at 100° C. for 2 hr, the mixture isfiltrated to form the first decoction. Further, 700 kg of water is addedfor extraction at 100° C. for 2 hr to form the second decoction. The twodecoctions are combined into a 1340 kg of decoction and concentratedunder reduced pressure to form a 42 kg of concentrate with 25 wt % solidcontent (the first concentrate). Then, the concentrate is stirred, and70 L of 95% ethanol is added for ethanol precipitation by using aperistaltic pump. The ethanol precipitate is separated by a centrifuge,and the supernatant is collected, concentrated under reduced pressure toform a 12.5 kg concentrate with 15 wt % solid content (the secondconcentrate). The solid phase and liquid phase of the second concentrateare separated by a centrifuge, and the supernatant is collected andpassed through a HP20 resin column (macroporous, styrene serialadsorption/desorption resin), and then washed with 30 L of water. Aftereluting with 30 L of 50% ethanol (95% ethanol/water=1V/1V), a 29.1 kg of50% ethanol elution fraction is obtained. Moreover, a 12.3 kg of ethanolelution fraction is obtained after eluting with 15 L of ethanol (95%ethanol). The 50% ethanol elution fraction and the ethanol elutionfraction are collected and combined, and concentrated under reducedpressure to form a 1.9 kg concentrate with 21.6 wt % solid content (thethird concentrate). The lyophilized product is 0.415 kg in weight, andcoded as BMEC-101.

Embodiment 4 Comparison of the Purification Factor of Each Process

In Table 1, to begin with 100 kg of botanical raw material, thepurification factor is increased up to 200 for BMEC-101 with thosesequencial purification procedures from extraction, ethanolicpurification and resin purification. TABLE 1 The relationship of productpurification factors Botanical raw Product material JM BMEC-1 BMEC-101Weight (kg) 100 10 5 0.5 Purification 1 10 20 200 FactorPurification factor =Weight_(botanical raw material)/Weight_(purified product JM, BMEC-1 or BMEC-101)

Embodiment 5 Animal Test (In Vivo)—d-Galactosamine-Induced AcuteHepatitis (the Positive Control Group is Orally Given with Guanine)

Male rats are randomly divided, with five rats in the individual groupand 200±20 g of each. In the study, normal and d-galactosamine(Gal)groups are orally given with distilled water, testing groups are orallygiven with test articles (JM or BMEC-1 from different processes)dissolving in distilled water, positive control group is orally givenwith guanine (300 mg/kg), and each administration dosage is 10 ml/kg.

Half-hour later, each group is i.p. with d-galactosamine (500 mg/kg)except the normal group. Four and eight hours after d-galactosamineinjection, the same dosages are administrated again. Twenty-four hoursafter d-galactosamine injection, the animals are sacrificed forcollecting the blood, and then the serum GOT (Glutamyl OxaloaceticTransaminase) and GPT (Glutamyl Pyrubic Transaminase) activities aremeasured by using HITACHI auto-analyzing system (model 7050) with UVmethod.

Embodiment 6 Animal Test (In Vivo)—D-Galactosamine-Induced AcuteHepatitis (Two Positive Control Groups are Orally Given with Silymarinand Guanine, Individually)

Rats are randomly divided, with six rats in each group. The animalsfasted for 24 hr before the experiment. In the study, normal andd-galactosamine (Gal) groups are orally given with distilled water,testing groups are orally given with 1 g/kg of the test articles (JM,BMEC-1 or BMEC-101 from different processes) dissolving in distilledwater, and rats in positive control group are orally administrated withsilymarin (200 mg/kg) and guanine (300 mg/kg). One hour later, eachgroup is i.p. with d-galactosamine (400 mg/kg), and the normal group isi.p. with saline. Four and eight hours after d-galactosamine injection,the same dosages are administrated again. Twenty-four hours afterd-galactosamine injection, the animals are anesthetized with ether, andthe blood is collected from the carotid artery. The serum is separatedand stands still at room temperature for 1 hr, and centrifuged (Backmancentrifuge, GS-6R, 3000 rpm) for 10 mins. The activities of rat serumGOT and GPT are measured.

Embodiment 7 Preparation of the Histopathological Sections

After blood is collected, the liver of the acute hepatitis animalinduced by d-galactosamine is separated, and liver tissue around 0.5 cm³in each folium is taken out. Those tissues are fixed in 10% neutralformalin for 1 to 2 weeks, then dehydrated and paraffin embedded by thedehydration and wax-exudation device, and cut into 4 to 5 μm liverslices by rotary microtome. The slices are stained with Haematoxylin andEosin, and the pathological patterns are observed under an opticalmicroscope.

Embodiment 8 Experimental Results

When the hepatocytes are damaged, liver enzymes will be largely releasedto the blood stream whereby the serum GOT and GPT activities areelevated. Thus, mouse serum GOT and GPT variations can be comparedbefore and after Boehmeria frutescens Thunberg extract treatment, tospeculate the repairing effect of Boehmeria frutescens Thunberg extracton liver damage. Also, liver weights are compared to evaluate the liverswell situation. The results are as follows:

(a) Table 2 Shows the Hepaprotective Effect of Boehmeria frutescensThunberg Extract (JM) on the d-Galactosamine-Induced Acute Hepatitis inRats TABLE 2 The effect of Boehmeria frutescens Thunberg extract (JM) onthe d-galactosamine - induced acute hepatitis in rats Dosage sGOT sGPTGroups (mg/kg) (U/L) (U/L) Normal —  180.0 ± 24.2  84.8 ± 8.9d-galactosamine 500 1298.0 ± 57.3 871.2 ± 58.9 (Gal) Gal+ guanine  300 ×3  980.0 ± 92.5*** 589.6 ± 54.6*** Gal + JM 1000 × 3  849.6 ± 202.6**396.0 ± 59.2***(Sample n = 5, value is represented as mean ± SEM, and compared with thed-galactosamine group with significant difference indicated by *p <0.05,# **p < 0.01, and ***p < 0.001 with student's t-test analysis.)

High serum GOT and GPT activities induced by d-galactosamine are reducedsignificantly after Boehmeria frutescens Thunberg extract (JM) treatment(the reduction of sGOT and sGPT values are 35% and 55%, individually).The positive control group is compared (orally given with guanine, andsGOT and sGPT values are reduced 24% and 32%, individually) to evaluatethe protection or repairing function of JM in the liver damage inducedby d-galactosamine.

(b) Table 3 shows the Hepaprotective Effect of Boehmeria frutescensThunberg Extract (BMEC-1) on the d-Galactosamine-Induced Acute Hepatitisin Rats TABLE 3 The effect of Boehmeria frutescens Thunberg extract(BMEC-1) on the d-galactosamine - induced acute hepatitis in rats DosageGroup (mg/kg) sGOT (U/L) sGPT (U/L) Normal —  110.4 ± 8.2  39.6 ± 1.3d-galactosamine 500 1727.6 ± 182.9 1255.2 ± 125.1 (Gal) Gal+ guanine 300 × 3  954.8 ± 122.1***  514.4 ± 78.2*** Gal + BMEC-1 1000 × 3  618.4± 102.8***  414.0 ± 67.6***(Sample n = 5, value is represented as mean ± SEM, and compared with thed-galactosamine group with significant difference indicated by *p <0.05,# **p < 0.01, and ***p < 0.001 with student's t-test analysis.)

After orally administrating 1000×3 mg/kg Boehmeria frutescens Thunbergextract (BMEC-1) prepared from the Embodiment 2, the high serum GOT andGPT values induced by d-galactosamine are significantly reduced (thereduction of sGOT and sGPT values are 64% and 67%, individually). Thepositive control group is compared (orally given 300×3 mg/kg guanine,and sGOT and sGPT values are reduced by 45% and 59%, individually) toevaluate the protection and repairing functions of BMEC-1 in preventingd-galactosamine-induced liver damage.

(c) Table 4 shows the Effects of Different Batches of Boehmeriafrutescens Thunberg Extract (BMEC-1) and Dose Dependence on thed-Galactosamine-Induced Acute Hepatitis in Rats

To understand the reproducibility of BMEC-1 preparation process from theEmbodiment 2, two continuous batches were produced using the processfrom the Embodiment 2. The consistency of the two batches is observed,and the protection efficacy of d-galactosamine-induced acute hepatitiswith treatment of different dosages is examined. The result is describedin Table 4. TABLE 4 Effects of different batches of Boehmeria frutescensThunberg extract (BMEC-1) and dose dependence effect on thed-galactosamine - induced acute hepatitis in rats Dosage sGOT sGPT Batch1 (mg/kg) (U/L) (U/L) Normal —  140.8 ± 11.6  41.2 ± 3.2 d-galactosamine500 2054.8 ± 227.9 1187.6 ± 156.8 (Gal) Gal+ guanine  300 × 3 1055.6 ±150.1***  699.2 ± 103.6*** Gal + BMEC-1 1000 × 3  887.6 ± 120.1*** 478.4 ± 70.4*** Gal + BMEC-1  300 × 3 1132.4 ± 143.0***  780.8 ± 80.2**Gal + BMEC-1  100 × 3 1623.2 ± 165.5**  906.4 ± 73.4* Dosage GOT GPTBatch 2 (mg/kg) (U/L) (U/L) Normal —  123.6 ± 4.7  32.0 ± 2.8d-galactosamine 500 1872.8 ± 246.6 1137.2 ± 125.4 (Gal) Gal+ guanine 300 × 3 1080.0 ± 181.7***  584.0 ± 114.9*** Gal + BMEC-1 1000 × 3 966.4 ± 151.6***  525.6 ± 101.1*** Gal + BMEC-1  300 × 3 1238.0 ±164.1**  646.8 ± 86.5** Gal + BMEC-1  100 × 3 1645.2 ± 185.6  995.6 ±117.7(Sample n = 5, value is represented as mean ± SEM, and compared with thed-galactosamine group with significant difference indicated by *p <0.05,# **p < 0.01, and ***p < 0.001 with student's t-test analysis.)

After orally administrating (1000×3 mg/kg) Boehmeria frutescens Thunbergextract (coded BMEC-1) prepared from the Embodiment 2 (Batch 1), thehigh serum GOT and GPT values induced by d-galactosamine aresignificantly reduced (the reduction of sGOT and sGPT values are 57% and60%, individually). The positive control group is compared (orally given300×3 mg/kg guanine, and sGOT and sGPT values are reduced by 49% and41%, individually).

Similarly, after orally administrating (1000×3 mg/kg) with Boehmeriafrutescens Thunberg extract (coded BMEC-1) prepared from the Embodiment2 (Batch 2), the high serum GOT and GPT values induced byd-galactosamine are significantly reduced (the reduction of sGOT andsGPT values are 48% and 54%, individually). The positive control groupis compared (orally given 300×3 mg/kg guanine, and sGOT and sGPT valuesare reduced by 42% and 49%, individually).

Accordingly, for the liver protection and repairing, both batches of theethanol-precipitated supernatant of Boehmeria frutescens Thunbergextract (BMEC-1) can effectively prevent the d-galactosamine-inducedliver damage.

In the dose dependence evaluation study, the examined data of those twobatches both show a dose dependence decrease of serum sGOT and sGPT byBMEC-1 administration (three dosages: 100×3 mg/kg, 300×3 mg/kg, and1000×3 mg/kg).

(d) Table 5 Shows the Effect of Boehmeria frutescens Thunberg Extract(BMEC-101) Prepared from Further Purification Process (Embodiment 3) andDose Dependence Effect on the d-Galactosamine-Induced Acute Hepatitis inRats.

To effectively retain and concentrate the active components of Boehmeriafrutescens Thunberg extract, and to largely reduce the therapeutic dose,the present invention further modifies the process of the Embodiment 2into the Embodiment 3, to provide a more active composition. Theabove-mentioned active composition is used in the treatment ofd-galactosamine-induced acute hepatitis in rats, and the result is shownin Table 5. TABLE 5 The effect of Boehmeria frutescens Thunberg extract(BMEC-101) prepared from further purification process on thed-galactosamine - induced acute hepatitis in rats Dosage sGOT sGPT Group(mg/kg) (U/L) (U/L) Normal — 131.2 ± 2.0  43.6 ± 4.6 d-galactosamine(Gal) 400 528.8 ± 66.3 259.6 ± 42.2 Gal+ guanine 300 × 3 160.7 ± 33.5** 55.1 ± 19.2* Gal+ silymarin 200 × 3 153.8 ± 8.9***  31.1 ± 1.3** Gal +BMEC-1 500 × 3 280.9 ± 31.9 150.1 ± 19.8 Gal + BMEC-101  50 × 3 106.0 ±3.3***  37.2 ± 4.8**(Sample n = 6, value is represented as mean ± SEM, and compared with thed-galactosamine group with significant difference indicated by *p <0.05,# **p < 0.01, and ***p < 0.001, after Sheffe's test, the sample isanalyzed with one-way ANOVA.)

After orally administrating (50×3 mg/kg) product (BMEC-101), the highserum GOT and GPT values induced by d-galactosamine are significantlyreduced (the reduction of sGOT and sGPT values are 80% and 86%,individually). The positive control groups are compared (orallyadministrated 300×3 mg/kg guanine, and sGOT and sGPT values are reducedby 70% and 79%, individually; orally administrated 200×3 mg/kgsilymarin, and sGOT and sGPT values are reduced by 71% and 88%,individually). Accordingly, it is shown that the product (BMEC-101) ofthe present process of the Embodiment 3 can possess the liver protectionand repairing functions. Moreover, the therapeutic dose is only 50×3mg/kg compared with the 200×3 mg/kg silymarin, and the 300×3 mg/kgguanine as positive control, it is shown that BMEC-101 possess a betterliver protection potency than silymarin. It is also shown that BMEC-101is more potent in liver protection than BMEC-1 with much lower dosage.

(e) Result of Histopathological Examination TABLE 6 The report of thehistopathological sections Histopathological Silymarin Guanine BMEC-1BMEC-101 examination Normal Gal 200 mg/kg 300 mg/kg 500 mg/kg 50 mg/kgInflammation 1 3 1 1 2 1 Necrosis 0 3 0 0 2 0 Fatty change 0 1 1 1 1 1Balloon 0 0 0 0 0 0 degeneration Bile duct 0 3 2 1 2 2 proliferationMitosis 0 0 0 0 0 0 Fibrosis 0 2 1 1 1 1(The evaluation of liver damage: 0 = absent; 1 = trace; 2 = weak; 3 =moderate; 4 = strong.)(The evaluation of liver fibrosis: 0 = normal; 1 = proliferation ofcollagen fibrosis without forming a septum; 2 = central vein and portalzone are# separated; 3 = an intact septum is formed with a crossing in themiddle, and the liver is substantially separated into several fractionsbut the septum is # still thin; 4 = an intact septum is formed and theseptum becomes thick, liver cirrhosis.)

According to Table 6 and FIG. 1, it is shown that the negative controlgroup, d-galactosamine (Gal) induced liver damage, results in cellinflammation, cell necrosis and bile duct proliferation (FIG. 1 b).Also, a mild fatty change and a liver fibrosis in the middle of centralvein and portal zone are observed. In the comparison among the testgroups (BMEC-1 as FIG. 1 e, and BMEC-101 as FIG. 1 f), the positivecontrol group (silymarin as FIG. 1 c, and guanine as FIG. 1 d) and thenegative control group (d-galactosamine as FIG. 1 b), Table 6 and FIG. 1indicate that both BMEC-101 and BMEC-1 do not have damage in terms ofballooning degeneration and mitosis. Moreover, the therapeutic dose ofBMEC-101 is only 50 mg/kg, and compared with the silymarin (200 mg/kg)and the guanine (300 mg/kg), it is shown that BMEC-101 possesses abetter liver protection than that of the silymarin. Also, it is shownthat the active component is effectively retained and concentrated so asto obtain the desired effect. For the fatty change, liver fibrosis, cellinflammation, cell necrosis and bile duct proliferation, BMEC-101 andBMEC-1 are both identical to the positive control group. In thecomparison with the negative control group, only a mild or weak damageoccurs in both of them.

From the above results, it is known that the Boehmeria frutescensThunberg extract prepared from the present invention can significantlyreduce serum GOT and GPT activities, namely, repair liver damage.Moreover, a low dose can achieve the desired effect, which alsoindicates an effective retaining and concentration of the activecomponent from Boehmeria frutescens Thunberg extract. It is known thatthe preparation method of the present invention is not only a noveltybut also capable of largely extracting the effective substance, and thusable to completely keep it from losing the activity.

In addition, without using toxic organic solvents, the extraction methodof the present invention utilize ethanol and water only, which areharmless to the human body. And, those skilled in this art realize thatapplying the alcohol extraction can scarcely obtain extract with highpotency. However, with further studies and several experiments by theinventor, the alcohol concentrations used to extract maximum amounts ofeffective components are obtained, and the animal study also verifiestheir particular efficacy. The disclosure of the present art means anavoidance of the toxic solvents such as methanol, chloroform, or acetoneto obtain a desired product for the hepatitis therapy.

Although the present invention has been explained in relation to itspreferred embodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the invention as hereinafter claimed.

1. A preparing method of a pharmaceutical mixture for the treatment ofhepatitis, comprising the following steps: (a) pulverizing a plant,macerating and extracting the plant with water; (b) concentrating theaqueous extract as the first concentrate; (c) adding ethanol to saidfirst concentrate to produce a precipitate and to form the separatedsolid and liquid phase, and separating said solid phase and said liquidphase; and (d) collecting and concentrating said liquid phase to form asecond concentrate, wherein said plants are Boehmeria frutescensThunberg, Boehmeria nivea or the nettle family.
 2. The preparing methodas claimed in claim 1, wherein said macerating in step (a) is tomacerate said plant with water for 8-24 hr.
 3. The preparing method asclaimed in claim 1, wherein said step (a) comprises boiling and stirringsaid plant for at least once, and combining each extract to form saidaqueous extract.
 4. The preparing method as claimed in claim 1, whereinsaid step (b) is concentrating said aqueous extract till its solidcontent reaches 1-50 wt %.
 5. The preparing method as claimed in claim1, wherein the initial concentration of said ethanol in step (c) is 95%and its final mixture concentration is 40-80 wt %.
 6. The preparingmethod as claimed in claim 1, wherein said solid phase and said liquidphase of step (c) are separated with centrifugal filtration method. 7.The preparing method as claimed in claim 1, wherein said concentrationin step (d) is concentrating said filtrate till its solid contentreaches 5-50 wt %.
 8. The preparing method as claimed in claim 1,further comprising drying, pulverizing, granulating and encapsulatingsaid second concentrate.
 9. The preparing method as claimed in claim 8,wherein said drying method comprising lyophilizing, spray and granulatedrying, or fluidized-bed drying.
 10. The preparing method as claimed inclaim 1, wherein said step (d) is prior to a step (e), in which saidsecond concentrate is passed through macroporous resin or ion exchangeresin.
 11. The preparing method as claimed in claim 10, wherein saidstep (e) is prior to a step (f), sequentially eluting the elutionfraction of step (e) with water, water-ethanol mixture and ethanol. 12.The preparing method as claimed in claim 11, wherein said step (f) isprior to a step (g), collecting and combining the water-ethanol andethanol elution fraction.
 13. The preparing method as claimed in claim12, wherein said step (g) is prior to a step (h), concentrating saidcombined elution fraction to form a third concentrate.
 14. The preparingmethod as claimed in claim 13, wherein said step (h) is prior to a step(i), drying and pulverizing said third concentrate.
 15. The preparingmethod as Claimed in claim 14, wherein said drying method in step (i) islyophilizing, spray and granulating drying, or fluidized-bed drying. 16.A pharmaceutical composition for the treatment of hepatitis, prepared bythe following steps: (a) pulverizing a Boehmeria frutescens Thunberg,Boehmeria nivea or the nettle family, macerating with water for 8 to 24hr, and extracting to form an aqueous extract; (b) concentrating saidaqueous extract to form a first concentrate; (c) adding ethanol to saidfirst concentrate to produce a precipitate and to form the separatedsolid and liquid phases, and separating said solid phase and said liquidphase; and (d) concentrating said liquid phase to form a secondconcentrate, and then drying said second concentrate.
 17. Thepharmaceutical composition for the treatment of hepatitis as claimed inclaim 16, wherein said step (a) comprises boiling and stirring saidplant for at least once, and combining each extract to form said aqueousextract.
 18. The pharmaceutical composition for the treatment ofhepatitis as claimed in claim 16, wherein said step (b) comprisesconcentrating said aqueous extract till its solid content reaches 1-50wt %.
 19. The pharmaceutical composition for the treatment of hepatitisas claimed in claim 16, wherein the initial concentration of saidethanol in step (c) is 95% and its final mixture concentration is 40-80wt %.
 20. The pharmaceutical composition for the treatment of hepatitisas claimed in claim 16, wherein said solid phase and said liquid phaseof step (c) are separated with a centrifugal filtration method.
 21. Thepharmaceutical composition for the treatment of hepatitis as claimed inclaim 16, wherein said concentrating method in step (d) is concentratingsaid filtrate till its solid content reaches 5-50 wt %.
 22. Thepharmaceutical composition for the treatment of hepatitis as claimed inclaim 16, further comprising drying, pulverizing, granulating andencapsulating said second concentrate.
 23. The pharmaceuticalcomposition for the treatment of hepatitis as claimed in claim 16,wherein said step (d) is prior to a step (e), in which said secondconcentrate is passed through macroporous resin or ion exchange resin.24. The pharmaceutical composition for the treatment of hepatitis asclaimed in claim 23, wherein said step (e) is prior to a step (f),sequentially eluting the elution fraction of step (e) with water,water-ethanol mixture and ethanol.
 25. The pharmaceutical compositionfor the treatment of hepatitis as claimed in claim 24, wherein said step(f) is prior to a step (g), collecting and combining the water-ethanoland ethanol elution fraction.
 26. The pharmaceutical composition for thetreatment of hepatitis as claimed in claim 25, wherein said step (g) isprior to a step (h), concentrating said combined elution fraction toform a third concentrate.
 27. The pharmaceutical composition for thetreatment of hepatitis as claimed in claim 26, wherein said step (h) isprior to a step (i), drying and pulverizing said third concentrate. 28.The pharmaceutical composition for the treatment of hepatitis as claimedin claim 27, wherein said drying in step (i) is lyophilizing, spray andgranulating drying, or fluidized-bed drying.